AI-Enhanced MALDI-TOF MS Analysis for Important Peaks on Predicting Ciprofloxacin Resistance across Different Gram-Negative Bacteria

Rapid identification of antibiotic-resistant infections is crucial, as antimicrobial resistance is a global health crisis. Yet, conventional antibiotic susceptibility tests (AST) often require days to yield results. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, cost-effective tool for bacterial identification and shows promise for resistance profiling by detecting spectral biomarkers. In this study, we harness MALDI-TOF MS with machine learning and deep learning to predict ciprofloxacin resistance across four Gram-negative bacteria, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Acinetobacter nosocomialis, using a cross-species "basket-wise" approach. We extracted features from mass spectra using kernel density estimation-based peak detection and m/z binning, then trained a random forest (RF) classifier and a convolutional neural network (CNN) to distinguish ciprofloxacin-resistant and susceptible isolates. To interpret the models, we employed dual feature importance analyses: gradient-weighted class activation mapping (Grad-CAM) for the CNN to highlight critical m/z regions and an ensemble RF-based method to identify significant peak features. The CNN achieved higher overall accuracy than the RF, especially in three of four species, while the ensemble RF approach identified interpretable sets of around 20 important m/z peaks per organism. Several informative peaks overlapped between species, indicating some common resistance-associated spectral signatures. However, no single universal marker was found across all species. These findings demonstrate an AI-enhanced MALDI-TOF MS framework for rapid AMR detection, yielding accurate predictions and interpretable spectral markers. The approach highlights clinical potential to guide effective therapy and bolster antimicrobial stewardship, particularly for underrepresented pathogens such as A. nosocomialis.